The objectives of the proposed research are to characterize the adenylate cyclase and phosphodiesterase activities in parathyroid tissue with respect to the influence of calcium and magnesium on their activities and to investigate the possible role of the parathyroid calcium-binding protein as a regulator of one or both of these enzymatic activities. In addition, to facilitate studies on the parathyroid calcium-binding protein with respect to its tissue specificity, its association with specific subcellular fractions of parathyroid tissue, and investigation of other possible roles it may have in parathyroid calcium-binding protein and a radioimmunoassay for its measurement developed. For the enzymatic studies, bovine parathyroid tissue will be obtained free of fat-cell contamination by collagenase dispersion. Adenylate cyclase will be initially studied in membrane preparations of parathyroid tissue, but attempts will also be made to solubilize the adenylate cyclase using non-ionic detergents. To isolate the individual phosphodiesterases, extracts of parathyroid tissue will be subjected to gel filtration and ion-exchange chromatography. Purified bovine calcium-binding protein will be tested for its effects on both the adenylate cyclase and phosphodiesterase activities with respect to Km's for their respective substrates and sensitivity to calcium and magnesium. Calcium binding protein will be purified from bovine parathryoid glands by methods previously published by the PI.